Conventionally, the exploration of new drugs is based on screening various compounds through computer research, and the majority of screened compounds target proteins.
Unlike traditional drugs, nucleic acid drugs inhibit the expression of target-specific messenger RNA (mRNA), making it possible to address research areas in which diseases could not be treated by conventional drugs that target proteins (Kole R. et al., Nature Rev. Drug Discov. 2012; 11; 125-140, Wilson C. et al., Curr. Opin. Chem. Bio. 2006; 10: 607-614.).
Despite the excellent effects and various applications of gene expression regulation based on oligo-nucleic acids, there are a number of obstacles to overcome in the development of nucleic acid-based therapeutic agents. For example, oligo-nucleic acids are at risk of damage by nuclease and the like, and the passage of oligo-nucleic acids through the cell membrane by passive diffusion is impossible due to the electrical properties (charges) and size of these oligo-nucleic acids. To overcome these problems, efforts have been continuously made to ensure biological stability through nucleic acid modification. For modified artificial nucleic acids, it becomes possible to increase their affinity for target nucleic acids without loss of biological activity.
Peptide nucleic acid (PNA), a kind of modified artificial nucleic acid, is an artificial nucleic acid having a (2-aminoethyl)-glycine peptide backbone introduced therein, and has the property of strongly binding to RNA and DNA, each having a nucleotide sequence complementary thereto. In particular, the peptide nucleic acid is resistant to nuclease and has high biological stability, and thus studies on therapeutic agents based on various oligo-nucleic acids have been conducted. However, the peptide nucleic acid has a disadvantage in that it is difficult to introduce into cells, because it is electrically neutral in nature (Joergensen M. et al., Oligonucleotides 2011, 21; 29-37.).
Owing to the performance and advantages of nucleic acids as drugs, various clinical trials using nucleic acids are in progress. Despite the increasing applications of nucleic acid-based therapeutic acids, the use of carriers for intracellular introduction is extremely limited. For example, clinical trials have been performed using a strategy (method) that delivers oligo-nucleic acid-based drugs into cells or tissues by use of nanoparticles, cationic liposomes and polymeric nanoparticles. However, most of these clinical trials do not include delivery systems, and rely mainly on direct introduction of nucleic acids by parenteral administration routes, including intramuscular injection, intraocular administration, subcutaneous injection and the like.
In addition, the cell membrane permeability of oligo-nucleic acids themselves is considerably low, and in particular, DNA or RNA is negatively charged. For this reason, these oligo-nucleic acids cannot pass through the hydrophobic phospholipid bilayer of the cell membrane, and thus are difficult to deliver into cells through simple diffusion. The use of a virus carrier such as retrovirus or AAV (adeno-associated virus) makes it possible to introduce oligo-nucleic acids into cells, but has risks, such as unintended immune activity and the possible recombination of oncogenes (Couto L. B. et al., Curr. Opin. Pharmacol. 2010, 5; 534-542.).
For this reason, the development of nucleic acid carriers based on non-viral oligo-nucleic acids having low cytotoxicity and low immune activity is of increasing importance. As a result, techniques of introducing nucleic acids using cationic lipids, liposomes, stable nucleic acid lipid particles (SNALPs), polymers and cell-penetrating peptides have been developed (Zhi D. et al., Bioconjug. Chem. 2013, 24; 487-519, Buyens K. et al., J. Control Release, 2012, 158; 362-70, ROSSI, J. J. et al., Gene Ther. 2006, 13: 583-584, Yousefi A. et al., J. Control Release, 2013, 170; 209-18, Trabulo S. et al., Curr. Pharm. Des. 2013, 19; 2895-923.).
These nucleic acid delivery techniques have functional moieties by direct binding, include a complex formation step, and have problems associated with the endosomal escape efficiency of liposome structures, in vivo toxicity, and the like. Consequently, it is required to improve the function of introducing oligo-nucleic acids and overcome problems associated with production procedures and side effects.
Under this technical background, the present inventors have made extensive efforts to develop a new structure having low cytotoxicity, an ability to allow a bioactive nucleic acid to permeate into cells, and an increased ability to regulate gene expression, and as a result, have found that a nucleic acid complex comprising a bioactive nucleic acid complementarily bound to a carrier peptide nucleic acid modified to be generally positively charged has a surprisingly increased cell permeability, and expression of a target gene can be very efficiently regulated using the nucleic acid complex, thereby completing the present invention.